The present invention relates to methods of determining teratogenic potentials of compounds of unknown teratogenic effects. More particularly, the present invention relates to the field of determining such teratogenic potentials without testing these compounds in mammalian species. In particular, the present invention relates to the field of screening tests for predicting the teratogenic effects of compounds on developing systems so as to estimate their potential for producing adverse effects on human embryos.
In recent years, there has developed an increasing interest in preclinically evaluating drugs and food additives to determine their teratogenic potential. In the near future, the Toxic Substances Control Act will become operative, and will result in guidelines for testing which will consist of the evaluation of several areas of toxicity. Typically, such evaluations will result in a tier system composed of increasingly costly and time consuming studies as one moves through a particular tier sequence. The lowest level of a tier ideally consists of a single or small group of comparatively inexpensive tests to reveal the most flagrant of the bad actors, i.e., those having the most adverse effects. A basic screen should give few if any false negatives, i.e., indicate that a chemical is not a hazard when it actually is. A chemical being evaluated would not necessarily be examined in higher level tests if no adverse effects are evident in the initial screen for that particular toxic effect. If the initial screen were such that it gives a few false positives, it would still be of value in a tier system of safety evaluations. A substance initially indicated as a bad actor by the initial screen can clear its "reputation" at a higher level in the tier system by indicating that the evaluation by the screen was a false positive. A tier system of evaluation is a realistic and pragmatic approach to the problem of regulating environmental chemicals.
It is estimated that 50,000 to 70,000 different chemicals are already in the marketplace and that 200 to 400 new chemicals are produced each year. The tire system approach is an attempt to protect the population from adverse effects by permitting a large number of substances to be tested while keeping costs low enough not to hinder research and development by chemical and industrial manufacturers.
At the present time, the one tier system which lacks any kind of basic, initial screen is that for teratology. No rapid and inexpensive screening system of teratogenic potential has yet been disclosed. A manufacturer can thus establish that a particular chemical does not adversely affect embryonic development only by performing intermediate or higher level tier system studies for teratogensis and embryo-lethal effects. In order to serve forthcoming needs in such a manner, great numbers of trained people will be required. In the absence of substantial resource allocation, screening for teratogenic potential may lag behind other safety evaluations.
A developmental test applicable as a screen of environmental agents must detect agents to which the conceptus is uniquely susceptible. D. Karnofsky first stated the concept now established as Karnofsky's Law that "virtually" any substance is capable of adversely affecting the conceptus if given at a high enough dose level. In determining whether a particular chemical substance needs to be regulated on the basis of being of a developmental hazard, it is important to determine if the embryo is uniquely susceptible to the agent. Agents which are coaffective teratogens (adversely affecting the embryo but only at a dose level near that adversely affecting the adult) would not necessarily be regulated as a developmental hazard. In this instance, the regulatory level could be established on the basis of its toxicity to the adult. Inclusion of a safety factor would provide protection to the conceptus from a coaffective teratogen. Only non-coaffective teratogens (those adversely affecting the embryo at a level markedly below the dose needed to adversely affect the adult) would require regulation below that level affecting the adult.
According to current screening techniques, rats are the preferred species for determining the teratogenic potential of a suspected agent. At the present time, it is necessary to use several hundred rats (a majority of them pregnant), over about three months time and $50,000 to make a determination of the dose level producing adult toxicity and the dose level adversely affecting embryonic development.
One species which has been intensely investigated is the fresh water coelenterate Hydra attenuata. This species is readily grown in the laboratory and has been a favorate of developmental biologists, having been the subject of over 2,000 papers published since 1744. Of the many species of Hydra, H. attenuata is most ammenable to the studies in the laboratory as it is not complicated by associated organisms, such as algae. Since the usual method (Loomis and Lenhoff, '56) of growing H. attenuata is quite time consuming, a semi-automated system for their growth has been developed which is the topic of my co-pending patent application entitled "Method and Apparatus for Growing Hydrozoa," Ser. No. 119,658, filed Feb. 8, 1980.
Many methods are known for collecting and dissociating H. attenuata into their component cells. These methods date back over 100 years, however, the method developed by Gierer et al., "Regeneration of Hydra from Reaggregated Cells," Nature New Biology, 239: 98-105 (1972) is presently preferred. The dissociated cells are randomly packed by centrifugation and expelled into culture medium as pellets. Over the next 24 hours the medium is reduced in molarity until that necessary for adults is attained. The randomly associated cells of a pellet rapidly develop into two tissue layers with endodermal cells internally positioned, ectodermal cells externally positioned, and multipotent interstitial cells which differentiate into nematoblasts and nerve cells interspersed between them. (See, David and MacWilliams, "Regulation of the Self-Renewal Probability in Hydra Stem Cell Clones," Proc. Natl. Acad. Sci. U.S.A., 75: 886-890, (1977) and Yaross and Bode, "Regulation of Interstitial Cell Differentiation in Hydra attenuata, " J. Cell Sci. 34: 1-26, (1978)). A pellet of 50,000 or more cells (Gierer et al., "Regeneration of Hydra from Reaggregated Cells," Nature New Biology, 239: 98-105 (1972)) is needed to achieve 100% of the pellets surviving to form multi-attached attenuata which separate from one another becoming free standing and feeding adult animals within seven days.
In order for new adults to be formed the cells must achieve; survival, changes in cell size and shape (Gierer et al., "Physical Aspects of Tissue Evagination and Biological Form," Quarterly Reviews of Biophysics, 10: 529-593 (1977), and Webster and Hamilton, "Budding in Hydra: The Role of Cell Multiplication and Cell Movement in Bud Initiation," J. Embryol. Exp. Morph., 27: 301-316 (1972)), selective cell death (Gierer et al., "Regeneration of Hydra from Reaggregated Cells," Nature New Biology, 239: 98-105, (1972)), cells must become spatially oriented, must recognize neighbors and form specialized junctions (Filskie and Flower, "Junctional Structures in Hydra," J. Cell Sci., 23: 151-172, (1977) and Wakeford, "Cell Contact and Positional Communication in Hydra," J. Embryol. Exp. Morph., 54: 171-183, (1979)), form selective adhesive associations and migrate, (Webster and Hamiston, "Budding in Hydra: The Role of Cell Multiplication and Cell Movement in Bud Initiation," J. Embryol. Exp. Morph., 27: 301-316, (1972)), induce differentiation of other cells less differentiated than themselves (Browne, "The Production of New Hydranths in Hydra by the Insertion of Small Grafts," J. Exp. Zool., (1909); Lee and Campbell, "Development and Behavior of an Integeneric Chimera of Hydra (Pelmathohydra Oligactis Interstitial Cells: Hydra attenuata Epithelial Cells)," Biol. Bull., 157: 288-296, (1979); and Sugiyama and Fujisawa, "Genetic Analysis and Developmental Mechanisms in Hydra VII. Statistical Analysis of Developmental Morphological Characters and Cellular Compositions," Develop., 1 Growth and Differ., 21: 361-375, (1979)), form intercellular matrix (Epp et al., "Isolation and Observation of Tissue Layers in Hydra attenuata Pall (Cnidaria, Hydrozoa)," Trans. Amer. Micros. Soc., 98: 392-400, (1979)), be responsive to inductive stimuli and differentiate (Berking, "Control of Nerve Cell Formation from Multipotent Stem Cells in Hydra," J. Cell Sci., 40: 193-205, (1979); Berking and Gierer, "Analysis of Early Stages of Budding in Hydra by Means of an Endogenous Inhibitor," Wilhelm Roux's Archives, 182: 117-129, (1977); Bode et al., "Regulation of Interstitial Cell Differentiation in Hydra attenuata, " J. Cell Sci., 20: 29-46 (1976); Browne, "The Production of New Hydranths in Hydra by the Insertion of Small Grafts," J. Exp. Zool., 7: 1-24, (1909); Schaller, "Action of the Head Activator as a Growth Hormone in Hydra," Cell Differentiation, 5: 1-11, (1976a); and Schaller, "Action of the Head Activator on the Determination of Interstitial Cells in Hydra," Cell Differentiation, 5: 13-20, (1976b)), form cell-specific organelles and products (Lentz, "Fine Structural Changes in the Nervous System of the Regenerating Hydra," J. Exp. Zool., 159: 181-194, (1965)), undergo mitotic division and then differentiate (Burnett et al., "Regeneration of a Complete Hydra from a Single, Differentiated Somatic Cell Type" (Chapter 11) in Biology of Hydra, Ed. by: A. L. Burnett, Academic Press, (1973); and David and Campbell, "Cell Cycle Kinetics and Development of Hydra attenuata," J. Cell Sci., 11: 557-568, (1972)), form organ fields (Browne, "The Production of New Hydranths in Hydra by the Insertion of Small Grafts," J. Exp. Zool., 7: 1-24, (1909); Gierer et al., "Physical aspects of Tissue Evagination and Biological Form," Quarterly Reviews of Biophysics, 10: 529-593, (1977); and Otto and Campbell, "Budding in Hydra attenuata: Bud Stages and Fate Map," J. Exp. Zoology, 200: 417-427, (1977)), and regulate organ field size (Bode et al., "Quantitative Analysis of Cell Types During Growth and Morphogenesis in Hydra," Wilhelm Roux' Archiv., 171: 269-285, (1973) and Webster and Hamilton, "Budding in Hydra: The Role of Cell Multiplication and Cell Movement in Bud Initiation," J. Embryol. Exp. Morph., 27: 301-316, (1972)), and become associated into tissues (Davis, "Histological and Ultrastructural Studies of the Basal Disk of Hydra III. The Gastrodermis and The Mesoglea," Cell Tiss. Res., 162: 107-118, (1975)) capable of functioning as parts of an integrated, coordinated adult. This is essentially the same list of phenomena required of a zygote in becoming an embryo and then a fetus. It also encompasses all of the phenomena considered vulnerable to abnormality during the pathogenesis of a developmental abnormality (Wilson, Environment and Birth Defects, Academic Press, New York, p. 25, (1973)). It is not being implied that these phenomena are all the same as in higher forms. Perhaps the molecular mechanisms to achieve them however are more similar than different. The only basis for this rather interesting (though highly speculative) thought is that an agent held to disrupt microtubules (vinblastine) interferes with a reaggregate's activities during the time of most active cell migration and shape changing while an agent perturbing DNA synthesis (methotrexate) disrupts regeneration at the time of greatest cell multiplication.
These cell aggregations may be considered artificial "embryos" because the actual pellet stage only lasts half a day, by which time they become hollow, bilaminar spheres. By two days, tentacle buds are present which develop into tentacles by the end of day three. Hypostomal anlagen are present on day four, and by five days axises are present. The organisms then detach as independent small adults within one week. As used hereinafter, the term "embryo" will be used to include the entire time course and sequence of events ranging from the pellet form, through the development of tissues, into detached, independent small adults.
Hydra attenuata is not the only species to have been subjected to intensive research concerning its developmental characteristics. In particular, cellular slime molds, Dictyostelium discoideum and plants have received some attention as systems which may permit the prediction of teratogenic potential. See for example Solomon, E. P., E. M. Johnson and J. H. Gregg, (1964) "Multiple Forms of Enzymes in a Cellular Slime Mold During Morphogensis," Developmental Biology, Vol. 9, pp. 314-326. See also, Cereon, G. and E. M. Johnson, (1971) "Control of Protein Synthesis During the Development of Acetabularia," J. Embyol. Exp. Morph., 26: 323-338; and Johnson, E. M., (1975) "Organ and Tissue Specificity in Response to Teratogenic Insult," appearing in New Approaches to the Evaluation of Abnormal Embryonic Development, edited by D. Newbert and H. J. Merker, Georg Thiem Publishers, pp. 573-590. It has also been suggested to use chicken eggs, Xenopus, and various sponges as systems for the screening of substances to determine their teratogenic potential. These systems have not gained wide spread acceptance, because of their cost, lack of reliability, limited response potential, or other factors interferring with simple, reliable testing.